| Project/Area Number |
10640673
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
動物生理・代謝
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| Research Institution | Okazaki National Research Institutes |
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Principal Investigator |
OGAWA Kazuo Okazaki national Research Institutes, Institute for Basic Biology, Associate Professor, 基礎生物学研究所, 助教授 (30132731)
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| Co-Investigator(Kenkyū-buntansha) |
KAGAMI Osamu Institute for Basic Biology, Research Associate, 基礎私物学研究所, 特別協力研究員
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Motor protein / Dynein / P-loop / Motor domain / Bacul virus / ATPase activity / バキュロウィルス / バァキュロウイルス発現系 / 生物分子モーター |
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| Research Abstract |
Dynein is a very huge motor protein in contrast to myosin and kinesin. There are two isoforms of dyneins; axonemal and cytoplasmic ones. However, dyneins so far cloned have four P-loop (ATP-binding) motives in the midregion of the molecules. In the present work, I have determined the true ATPase activity site of outer arm dynein from sea urchin sperm axonemes by the recombinant protein expression.
In an expression of a series of deleted mutants in Baculo virus system, products were not extracted under the mild condition possibly because of an abortive folding of expressed mutants. To get soluble mutants, we have tried the dual expression of mutants and the other protein component contained in outer arm dynein. In situ dynein (heavy chain of outer arm dynein) associates with intermediate chain (IC1). Thus, we expressed simultaneously a series of mutants and IC1 in Baculo virus system.
ICI was flagged with 6xHis. Taking the in situ binding of dynein heavy chain and IC1 into consideration,
… More
mutants could be bound to the Ni-affinity resin via the interaction of them with IC1-6xHis. I confirmed that mutants were bound to the resin, although the percentages of bound and unbound ones were very low.
Mutant ranged in the four P-Loop motives showed an ATPase activity, though to greatly Lesser extents than in situ dynein. So, I prepared a series of smaller mutants containing only one P-Loop motif in the molecule to identify the ATPase site of dynein heavy chain and the significance of four P-Loop motives. Dual expression of them and ICI-6xIlis and ATPase assay demonstrated that only the mutant containing N-terminal first P-Loop (PI) showed ATPase activity. The other P-loop motif itself didn't show any ATPase activity.
From the present work, the first P-loop motif of dynein molecule seems responsible for ATPase activity of dynein. Although the solubility of mutants was improved by the dual expression, Low specific activity of mutants suggest the occurrence of still abortive folding of them. Less
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